Apparatus and method

ABSTRACT

An apparatus for investigating a molecule comprising a channel provided in a substrate, a metallic moiety capable of plasmon resonance which is associated with the channel in a position suitable for the electromagnetic field produced by the metallic moiety to interact with a molecule passing therethrough, means to induce a molecule to pass through the channel, means to induce surface plasmon resonance in the metallic moiety; and means to detect interaction between the electromagnetic field produced by the metallic moiety and a molecule passing through the channel. Methods of investigating molecules are also provided.

The present invention relates to apparatus and methods for investigatingmolecules, in particular biological molecules such as nucleic acids orproteins. The apparatus and methods are concerned with application ofnanoscale technology to the problems of rapid and detailed analysis ofthese molecules. In one aspect the present invention relates toapparatus and methods of sequencing nucleic acids.

The ability to rapidly sequence nucleic acids remains of paramountimportance in biology. Although the entire sequences of many genomes(including human) are now known, and many more are becoming available,the remains a need to obtain sequence data for many reasons. Currentsequencing technologies are primarily based on the Sanger (“dideoxy”)technique and are laborious and slow. Methods involving the detection ofthe incorporation of single nucleotides by a polymerase areintrinsically limited to the relatively low speed at which a polymerasecan synthesise a complementary DNA strand. Currently the maximumsequencing rate for a single sequencing device is approximately 1base/second; though an improvement over conventional Sanger sequencingtechniques, this is still very slow.

Methods through which DNA is sequenced via detecting changes of currentas DNA passes through a conducting pore have been proposed. Althoughsuch methods have a high theoretical maximum speed of detection, to daterelatively low detection rates have been achieved and it seems likelythat without extensive modification to the overall design or the use ofconducting labels, the limit of detection will remain low.

Nanotechnology, like biology, is an area which is rapidly developing andis leading to novel approaches and materials for the manipulation andanalysis of molecules. Nanotechnologists are now able to produce andmanipulate materials down to the sub-nanometer scale. This provides thepotential for the development of devices or techniques with new andpreviously unconsidered potential. However, while nanotechnology hasfound utility, for example, in the electronics industry, it has not yetbeen widely harnessed in the field of biology.

Plasmon resonance is a phenomenon whereby plasmons on a metallic surfaceare excited by light incident upon the surface. Plasmon resonance isused in a number of conventional analytical machines, such as Biacore®.Where plasmons on a planar surface are excited the term surface plasmonresonance (SPR) is used, and where a nanometer sized particle is excitedthe term localised surface plasmon resonance (LSPR) is often used todifferentiate this aspect of the phenomenon. When a small metallicparticle is irradiated by light, the oscillating electric field causesthe particle's electrons to oscillate coherently. The collectiveoscillation of these electrons is called the dipole plasmon resonance.The collective oscillation in the electron cloud produces an intense,highly localised electromagnetic field (EMF). In order to produce thesecollective oscillations, the correct excitation wavelength must be used.This wavelength will depend on both the size and shape of the metalparticle or nanostructure being excited.

It has been discovered that by using highly localised SPR achievableusing nanotechnological techniques, the structure of biologicalmolecules can be investigated at the nanometer scale.

According to the present invention there is provided an apparatus forinvestigating a molecule comprising:

-   -   at least one channel provided in a substrate, the channel being        suitable for the passage of the molecule therethrough;    -   at least one metallic moiety capable of plasmon resonance, the        at least one metallic moiety being associated with said channel        in a position suitable for the electromagnetic field produced by        the metallic moiety to interact with the molecule passing        therethrough;    -   means to induce a molecule to pass through the channel;    -   means to induce surface plasmon resonance in the metallic        moiety; and    -   means to detect interaction between the electromagnetic field        produced by the metallic moiety and a molecule passing through        the channel.

Suitably the apparatus comprises two reservoirs: a first reservoirsuitable to receive a sample comprising the molecule to be investigated,and a second reservoir suitable for the molecule to pass into once ithas passed through the channel. Suitably the two reservoirs areseparated from each other by the substrate, and the molecule must passthrough the at least one channel to pass from the first reservoir intothe second reservoir. Thus the substrate could be considered to be amembrane or diaphragm separating the two reservoirs.

Generally the dimensions of the channel should be suitable for amolecule to be investigated to pass through the channel, and such thatthe molecule is constrained to pass suitably close to the metallicmoiety such that an interaction between the molecule and the localisedEMF produced by the metallic moiety occurs. The channel should generallyalso have suitable dimensions such that only one molecule can passthrough the channel at a time.

Generally it is preferred that the channel is an aperture, though it ispossible that other forms of channels such as slots or grooves providedin a substrate may also be suitable.

The aperture may have any suitable shape, although a circular orapproximately circular aperture is envisaged as being most convenient.

The aperture (or channel) may have a diameter of from approximately 0.5nm to 100 nm preferably 50 nm or less, especially 30 nm or less.Although the term diameter is used, it should be understood that it isnot meant in a strict geometrical sense and may, for example, apply tothe smallest dimension of a non-circular aperture or channel.

Often small apertures in a substrate are referred to as “pores”, andthis term may be used throughout the specification and should beconstrued accordingly.

Where the molecule to be investigated is a nucleic acid, the smallestaperture through which single-stranded nucleic acid will pass isapproximately 1 nm. Double-stranded nucleic acids will pass through anaperture of approximately 2 nm or greater [see Heng J. B. et al.Biophysical Journal (2006), 90, 1098-1106].

Accordingly, where a single stranded nucleic acid is to be investigated,it is preferable that the aperture should be from 1 nm to 100 nm indiameter, preferably 1 nm to 30 nm, especially 1 to 10 nm, particularly1 to 5 nm. For double stranded nucleic acid the aperture should be from2 nm to 100 nm in diameter, preferably from 2 nm to 30 nm, especiallyfrom 2 nm to 4 nm.

The channel may be formed of any suitable substance. It may be organicor inorganic.

In one embodiment the aperture may be formed from an “organic” pore,i.e. a pore derived from a naturally occurring structure, for example aprotein having a pore such as α-haemolysin or the organic pore found inthe outer membrane of mitochondria.

Alternatively the aperture could be provided by a gel conventionallyused for the manipulation of nucleic acid or other biological molecules,such as agarose or polyacrylamide.

Alternatively, the aperture could be engineered in a substrate such as afilm formed from silicon nitride or other such material. See, forexample, Ho C. et al., PNAS (2005), 102(30) 10445-10450 which discussesmethods of forming nanometer diameter pores from silicon nitride.

The aperture could also be formed from a nanoporous material such asporous gold, a colloidal crystal, colloidal cavities, metal toroids etc.Netti et al. describe the formation of gold nanocavities and theirability to form surface plasmons [see Netti M. C. et al. AdvancedMaterials (2001), 13(18), 1368-1370]. Aizpurua O. et al. describe theformation of gold nanorings [see Aizpurua et al. Physical Review Letters(2003), 90(5), 057401-1-057401-4]. Lesuffleur et al. discuss goldnanopore arrays with enhanced plasmon resonance [Lesuffleur A. et al.,Applied Physics Letter (2007), 90, 243110]. Where the aperture is itselfformed from a metallic substance capable if exhibiting LSPR, then itwill be apparent that a separate metallic moiety may not be required.

Means for nano-engineering to produce a variety of suitable structuresfor the present invention are well known, but this is also a rapidlydeveloping field of technology. As such, it is expected that noveltechnologies and materials suitable for forming channels in a substrateas required by the present invention will be developed in the future;such technologies are within the scope of the present invention.

It is generally preferred that the apparatus comprises a plurality ofapertures passing through the substrate. The apertures could be arrangedin an array where the position of each aperture is controlled; this isparticularly convenient where nano-engineering is used to form aperturesin a substrate. Alternatively, the apertures could be scattered acrossthe substrate without control over their position.

The metallic moiety must be able to form surface plasmons and thus forma localised EMF in the vicinity of the moiety. Any material which issuitable to achieve this function is suitable for use in the metallicmoiety. Gold, silver, copper and aluminium metals have been found tohave desirable properties in terms of plasmon formation and propagation,and thus it is generally preferred that the metallic moiety is formedpredominantly or entirely from one or more of these metals.

Nanotechnology allows the formation of gold or silver particles inessentially any shape. Shape can have a substantial effect on the EMFproduced by a particle [see Mock J. J. et al., Journal of ChemicalPhysics, 116(15), 6755-6759].

A spherical or substantially spherical (e.g. a sphere or spheroid)particle has suitable properties and thus forms a suitable embodiment ofthe metallic moiety. It has been found that spherical metal particleshaving a diameter of from 50 nm to 150 nm in diameter are especiallysuitable due to high levels of fluorescence enhancement [see Nakamuraand Hayashi, Japanese Journal of Applied Physics, 44(9A), 6833-6837].

In an alternative embodiment an annular metallic moiety may be used.Such an annular moiety has certain benefits; it has been shown that theEMF within a ring is strong and is relatively constant across the innerarea defined by the ring [see Aizpurua et. al., ibid]. Where themetallic moiety is a ring, it will be obvious that the ring could itselfform the aperture through which the molecule will pass. Methods ofmanufacturing such nano-rings are set out in Aizpurua et al.

Other shapes such as polygonal prism or other multi-sided shapes mayalso be suitable.

For the sake of clarity it is pointed out that there may be more thanone metallic moiety associate with a particular channel, and that morethan one type of metal may be used. Indeed, it may be desirable to usetwo or more metallic materials such that a region of maximal EMF isproduced as a result of combination of the separable EMFs of eachmoiety. This may give rise to extremely high EMF values in highlylocalised areas.

It is generally important that the metallic moiety is arranged in aposition such that it will interact with a molecule passing through thepore, but is spaced sufficiently from the pore to prevent or mitigatequenching of a signal emitted from the molecule. This can be achieved ina number of ways. The channel may be shaped and sized such that amolecule passing through it is constrained in a position such that itdoes not get too close (thus avoiding quenching), or too far away (thusensuring interaction) from the metallic moiety—this is especiallysuitable when the pore itself has no quenching activity. Alternatively,control means may be used to guide the molecule such that it does notget too close to the metallic moiety.

Typically the surface of the metallic moiety should be positioned to be50 nm or less from the molecule as it passes through the channel,preferably 30 nm or less, especially 10 nm or less.

The means to induce a molecule to pass through the channel may be one ormore of means to induce electrophoresis, a micro-fluidics device, or alaser trapping device. However, any other means suitable to induce themolecule to move to and/or through the channel may also be suitable.

The apparatus may comprise control means to control the passage of themolecule towards, into and/or through the aperture. The control meansmay be adapted to control one or more of the velocity, direction,position or conformation of the molecule.

The type of control means will vary depending on the type of molecule tobe investigated and the type of control required. Where sequencing ofnucleic acid is required, it is important that the speed of passage ofthe linear nucleic acid molecule through the channel is controlled. Forsequencing it is also desirable that the nucleic acid molecule is heldin a linear conformation.

In one embodiment the control means may comprise a porous matrix throughwhich the molecule passes, the porous matrix lying immediately adjacentto the entrance to the channel. Where a porous matrix is used, it isconvenient if the molecule is induced to pass through the matrix viaelectrophoresis. The properties of the porous matrix in combination withthe electrophoresis conditions serve to allow fine control of themovement of the molecule (e.g. nucleic acid) through the matrix and intothe channel. Accurate control of the speed of movement of a nucleic acidthrough a gel can be achieved. An additional advantage ofelectrophoresis though a porous matrix is that the conformation of alinear molecule is drawn out into an elongated, linear form which isparticularly suitable for sequencing. A suitable porous matrix may beformed from convention materials used for manipulating nucleic acids andproteins, such a polyacrylamide or agarose. Alternatively, othernano-porous materials may also be suitable.

The control means described above is well suited for the control ofnucleic acids and other linear molecules. Other molecules may require adifferent level of control, or the degree of control may be higher orlower.

It should be noted that the means to induce a molecule to pass throughthe channel and the control means may be provided by the same means. Forexample electrophoresis in a liquid medium alone may be suitable forcontrolling molecules where accurate control of conformation andvelocity is not required. Alternatively, laser trapping may be used toaccurately control the movement of a molecule toward and into thechannel.

The means to detect interaction of the metallic moiety and the moleculemay of course be any suitable device which can detect the interactionbetween the localised EMF produced by the moiety and the molecule inclose proximity to it. Suitable means may include means to detectfluorescence or means to detect Raman scattering.

Suitable means to detect fluorescence include any device which issensitive enough to detect photons emitted from a fluorophore passingthrough the region of EMF. Suitable devices include a photomultiplier oran avalanche photodiode. Fluorescence is suitable to detect inherentlyfluorescent molecules or molecules which have been labelled with afluorophore.

Suitable means to detect Raman scattering include a Raman spectrometer.Raman scattering may be used to detect unlabelled and non-fluorescentmolecules. Techniques to detect Raman scattering are well known in theart. Detection using Raman scattering in the present invention are basedon the phenomenon of surface enhanced Raman spectroscopy [see forbackground Jeanmaire, David L.; Richard P. van Duyne (1977). “SurfaceRaman Electrochemistry Part I. Heterocyclic, Aromatic and AliphaticAmines Adsorbed on the Anodized Silver Electrode”. Journal ofElectroanalytical Chemistry 84: 1-20.]

In many embodiments it may be preferred if a method of detection is usedwhich does not rely on labelling of the molecule. This simplifies thepreparation of the sample to be investigated, and also does not requirethe presence of a label which may interfere with the passage of themolecule through the channel.

In a further aspect the present invention provides a method forinvestigating a molecule comprising the steps of;

-   -   providing a fluid sample comprising a molecule to be        investigated;    -   inducing said molecule to pass through a channel provided in a        substrate, the channel having an associated metallic moiety        which is induced to have plasmon resonance;    -   detecting interaction between the electromagnetic field produced        by the metallic moiety and the molecule as it passes through the        channel.

Preferably the molecule is a biological molecule, e.g. a nucleic acid ora protein/peptide. Linear molecules such as nucleic acids or linearproteins/peptides are particularly suitable for investigation using thepresent method. Single-stranded nucleic acid is especially suited forinvestigation using the present method.

In a preferred embodiment the method is a method of sequencing a nucleicacid.

However, the method may also be used for investigation of other sorts ofmolecules, and need not be used for sequencing. The use of the methodfor counting individual molecules in a sample, and mapping of markers onlinear molecules are envisaged as being amongst other potential uses.

It may be preferred, though it is not essential, that the moleculecomprises a label which interacts with the metallic moiety. Accordingly,the method may further comprise the step of labelling the molecule witha label which will interact with said metallic moiety.

Suitable labels include fluorophores. Fluorophores are well known in theart in relation to fluorescent labelling of all types of biologicalmolecules. In the present invention it is important that the fluorophoreis small enough that it does not inhibit passage of the labelledmolecule through the channel. Typically the fluorophore will have aeffective size of 2 nm or less, and preferably a dye molecule measuringless than 1 nm in any dimension.

Examples of suitable fluorophores include Rose Bengal, Alexa Fluor 488C₅-maleimide, Alexa Fluor 532 C₂-maleimide and Rhodamine RedC₂-meleimide, and Rhodamine Green. These fluorophores can each be usedto label the bases of nucleic acids using techniques known in the art.

Suitably the molecule is a nucleic acid in which a substantialproportion or all of a particular base has been labelled. In a preferredembodiment a substantial proportion or all of one or more bases in themolecule are labelled, each type of base having a label with anindividually identifiable emission spectrum. In an especially preferredembodiment all, or substantially all the bases are labelled, each typeof base having a label with an individually identifiable emissionspectrum. High density labelling of nucleic acid is discussed in Tasaraet al., Nucleic Acids Research, 2003, 31(10), 2636-2646.

It is important that a fluorophore selected as a label is capable ofinteracting with the EMF produced by the metallic moiety. It is aroutine matter to match a fluorophore to a metallic moiety of particularproperties. In general for good interaction between the metallic moietyand the fluorophore, it is preferred that the fluorophore has anemission peak at slightly lower energy than the localised surfaceplasmon resonance peak of the metallic moiety, i.e. the fluorophoreemission peak is slightly red shifted from the LSPR peak. Red shifts offrom approximately 20 to 150 meV are suitable, preferably fromapproximately 40 to 120 meV [see Chen et al., Nano Letters (2007), 7(3),690-696].

According to a further aspect, the present invention provides asubstrate suitable for use in an apparatus for investigating a moleculecomprising:

-   -   at least one channel provided in the substrate, the channel        suitable for the passage of a molecule therethrough; and    -   at least one metallic moiety suitable to have plasmon resonance        induced therein, the at least one metallic moiety being        associated with said channel in a position such that the        electromagnetic field produced by the metallic moiety is able to        interact with a molecule passing therethrough.

Further details of preferred substrates are set out above.

Embodiments of the invention will now be described, by way ofnon-limiting examples, with reference to the accompanying drawings inwhich:

FIG. 1 shows a pore and electrode suitable for use in the presentinvention;

FIG. 2 shows a substrate comprising pores and electrodes;

FIG. 3 shows a schematic representation of aspects of an apparatusaccording to the present invention;

FIG. 4 shows a schematic representation of an embodiment of an apparatusaccording to the present invention; and

FIGS. 5 to 7 show schematic representations of alternative structures ofthe substrate.

BACKGROUND

When a small spherical metallic particle is irradiated by light, theoscillating electric field causes the particle's electrons to oscillatecoherently. The collective oscillation of these electrons is called thedipole plasmon resonance. The collective oscillation in the electroncloud produces an intense, highly localised electromagnetic field (EMF).In order to produce these collective oscillations, the correctexcitation wavelength must be used. This wavelength will depend on boththe size and shape of the metal particle being excited. The correctwavelength for a particular particle can easily be determinedexperimentally or, for certain shapes, may be predicted using knownmodeling techniques.

It has been shown in [1] that the EMF produced by the plasmon resonanceof metal colloids such as gold or silver nanoparticles can be used toexcite commonly used fluorescent labels such as Cy5 and Rose Bengal,amongst others. These experiments involved placing gold or silvernanoparticles on a surface and positioning the fluorophores such thatthey benefit from the enhanced EMF. This results in what is called metalenhanced fluorescence (MEF).

When a fluorophore is placed near a metal particle exhibiting plasmonresonance, the fluorophore benefits from both the localised EMF and froma less-well understood phenomenon in which the fluorescent lifetime isdecreased. A similar phenomenon occurs when the Raman scattering of amolecule is examined. Where the molecule if in close proximity to ametallic particle exhibiting localised surface plasmon resonance adramatic increase in the level of Raman scattering is observed.

As the maximum photon emission of a fluorophore is determined by itsquantum yield and fluorescent lifetime, a shorter fluorescent lifetimeresults in higher photon emission. A fluorophore having a 90% quantumyield and a fluorescent lifetime of 2.5 ns could emit a maximum of 360million photons/s. In reality, fluorescent dyes can emit several millionphotons before deteriorating.

By inducing a highly localised enhanced EMF, such as has beendemonstrated using gold and silver colloidal arrays, [1,2] as well as incolloidal nanocrystals [3], photonic crystal nanocavities [4], metalnanorings [5] and porous thin metal films [6] and positioning afluorophore such that it will benefit from it, MEF can be induced andthe fluorophore detected, or alternatively Raman scattering detected.

If a device is made such that there is a region in space where only onefluorophore may benefit from MEF at any given time, the position of thatfluorophore can be known. By arrangement of a plasmonic structure (e.g.a metallic moiety) around or adjacent to an aperture (pore), afluorescent particle can be oriented such that it passes through an areaof maximum fluorescent enhancement (MaxFE). Suitable pores include: gels(commonly used to confine polynucleotides), organic pores suchα-haemolysin, solid state pores such as can be made in silicon nitride,nanoporous materials such as porous gold, colloidal crystals, colloidalcavities, metal toroids, or any other pore less than 10 nm in diameterand preferentially less than 5 nm in diameter, especially between 1.5and 2.5 nm.

Given the following:

-   -   the EMF between two plasmon resonating surfaces increases when        the distance between them decreases,    -   that proximity to a plasmon resonating surface also diminishes        the fluorescent lifetime of the fluorophore,    -   that the distances between these surfaces can be made less than        5 nm,    -   and that colloids, porous thin films, 2D crystal arrays, and        other nanostructures can made less than 2 nm in size,        it is reasonable to assume that maximum photon emission can be        made to occur in an extremely small region.

In a similar way, particular properties of a molecule, or regions withina molecule, can be identified by characteristic patterns of Ramanscattering. This, as with the identification of the location of aparticular fluorophore, the position of a particular feature having anidentifiable Raman spectrum can be identified by its proximity to theplasmonic structure.

If an ion is induced to pass through a pore by means of, for examplemicrofluidics or electrophoresis, it can be made to pass through thisarea of MaxFE such that detection of a single ion-fluorophore conjugate,or a region having a particular Raman spectrum can be detected.

By inducing a fluorescently labelled charged ion such as a protein topass through a nanopore such that only one ion may travel through thepore at once, quantification of those ions would be possible as follows:

-   -   Given two identical fluorescent particle/ion conjugates, as the        first passes through the pore and enters the confined EMF, the        fluorophore will begin to emit photons. As the fluorophore moves        closer to the region of MaxFE, an increasing number of        fluorophores are emitted. As photon emission is directly related        to the intensity of the EMF, which in turn increases        exponentially as the distance to the region of MaxFE decreases,        the photon emission will also increase exponentially, producing        a photon emission peak in the region of MaxFE and decreasing as        the conjugate moves away from the area of MaxFE. The second        identical fluorescent particle will produce the same pattern of        photon emission. So long as peak photon emission occurs in a        discrete region with a thickness less than the distance between        the two fluorophore/ion conjugates, the fluorescence produced by        these two fluorophores can be differentiated.    -   Should the two conjugates be spaced so closely together (less        than 0.5 nm) that individual detection would be difficult, the        presence of more than one fluorophore in the area of MaxFE could        be determined by photon emission. Two identical fluorophores        exposed to equal EMF strengths should emit roughly twice as many        photons/s as a single fluorophore. Given that a region of MaxFE        can contain two, three or more conjugates, if the speed at which        they travel through the EMF is known, quantification of the        number of conjugates which pass through the EMF can be achieved.

By creating two chambers separated by a barrier which ensures that ionscould only pass from one side to the other via a pore, the ions whichcan be induced to migrate could be quantified regardless of whether theregion of MaxFE can contain two, three or more conjugates so long asthat number has been determined.

Labelling of Nucleic Acids

Fluorescent labels are commonly incorporated into the both singlestranded (ss) and double stranded (ds) polynucleotides DNA and RNA.There are many well-established methods for incorporating fluorescentlabels into DNA and by careful choice of both DNA polymerase andfluorescently modified nucleotide, it has been shown [7] that it ispossible to fluorescently label all of one or more of the four bases(CGTA). The distances between nucleotides in dsDNA and dsRNA is known tobe 0.34 nm, while in ssDNA and ssRNA, the polymers can be in linearconformation, allowing the maximum spatial separation to begreater—although the exact distance varies between 0.5 and 1 nmdepending on the stretching force imposed [8].

The Sequencing Process

It is known that precise control of the speed at which a single strandof DNA passes through an organic pore such α-haemolysin [10] or asolid-state pore in silicon nitride can be achieved [8,9]. By labellinga polynucleotide such that all of one or more of its bases arefluorescently labelled and inducing that polynucleotide bymicrofluidics, electrophoresis or some other means to pass through anEMF at a known speed such that the nucleotides pass through a region ofMaxFE able to contain at least one fluorescently labelled nucleotide,the position of the labelled nucleotides along the DNA fragment can bedetermined.

A complete polynucleotide sequence could be assembled in the followingway:

Assuming all of one base will be labelled at a time, four PCR reactionscan be performed in which in each, all of the C, T, G, or A nucleotidebases are fluorescently labelled. The strands are then denatured-formingssDNA or ssRNA. Each strand is then induced to pass through a nanoporeat a known speed and through an area of MaxFE able to contain a knownnumber of nucleotides. As the labelled nucleotides pass through the EMF,their positions can be determined relative to the other labellednucleotides—

-   -   providing a distance map for each of the four identical but        differently labelled polynucleotides. These four distance maps        can then be easily assembled into a complete polynucleotide        sequence.        Whole Genome Sequencing:

In order to sequence a genome using this method, fluorescently labeledrandom primers, such as are available for whole genome amplification,could be used. The sequence of these primers would be known and thefluorescent label could serve to indicate the 5′ or 3′ orientation ofthe DNA fragment as it passes through the pore. The labelled primerwould also serve as an initial reference point from which the distancemap of the labelled nucleotides could be related. Preferentially, all ofmore than one of the four bases would be fluorescently labeled andfluorescent labels with different emission spectra would be used todifferentiate these bases. More than one fluorescent label spectra couldalso be used to label all of one type of base providing the labels haddistinct max emission spectra.

These DNA fragments could then be induced to pass through the pore asdescribed above at a rate determined by the photon emission. Using thedye Cy5, photon emission rates for MEF of between 3 and 9 millionphotons/s have been obtained [1]. This could allow DNA to pass throughat a rate of slightly less than one million nucleotide bases per secondper nanostructure. An array of nanostructures would allow this speed tobe multiplied by the size of the array. As these primers are random andthe position of the distance maps for the DNA fragments within thegenome is not known, whole genome assembly software such as is used forwhole shotgun sequencing would be required for accurate alignment andassembly.

ADVANTAGES OF THE PRESENT INVENTION

The present invention provides a variety of advantages over othermethods of DNA sequencing as well as other methods of single moleculedetection:

-   -   The invention allows enhanced single molecule detection.    -   The invention can be used to determine the position and speed of        single molecules.    -   Although PCR can be used to fluorescently label single        molecules, the present apparatus and method is not reliant on        chemical or enzymatic reactions.    -   The apparatus can be arrayed such that tens, hundreds, thousands        or millions of these nanostructures can be in operation at once.    -   DNA sequencing using the method outlined above would be        extremely fast. The cost of sequencing using this method would        be limited to the cost of no more than four PCR reagents such as        fluorescent labels, nucleotides, polymerases and other minor        consumables.        Methods of Construction

The heart of the invention lies in inducing a single molecule to passthrough a highly localised EMF in a controlled manner and detectinginteraction between the molecule and the EMF. There are manywell-researched methods of controlling both proteins and polynucleotidesincluding gels, organic pores, solid state pores, attachment to beads,and immobilisation on a surface. This allows a wide range of devices tobe constructed which could control a single molecule such as a proteinor a DNA fragment such that it could be made to pass through a highlylocalised EMF.

The present invention allows any surface to be used so long as theplasmon resonance induced by an incident EMF will induce MEF in afluorescently labeled single molecule or SERS, and that single moleculecan be induced to pass through an area of MaxFE.

The present invention allows any means of inducing a single molecule topass through a region of MaxFE including microfluidics, electrophoresis,or laser trapping.

Where fluorescence is the chosen method of detection, the presentinvention allows any fluorescent label to be used which will fluoresceat the frequency of maximum plasmon resonance of the surface being used.

Ideally, the fluorescent label will have a high quantum yield, lowfluorescent lifetime, low molecular weight, and be easily incorporatedby a polymerase. Preferably, the nucleotides themselves areintrinsically fluorescent.

SERS has the advantage that no labeling is required, but rather thespecific Raman spectrum of features of the molecule to be identifiedshould be known.

These characteristics allow for the invention to be engineered in atleast three general ways, each of which having distinct advantages:

-   -   The use of an “organic” pore to control a single molecule allows        gold nanoparticles to be covalently bound either to the pore        itself or to the surrounding lipids. Alternatively, the organic        pore can be placed inside a larger solid state nanopore such as        can be made in silicon nitride, and the plasmonic structure        (e.g. metallic moiety) positioned such that MEF or SERS is        optimized for those molecules or parts of molecules within the        organic pore. Alternatively, an organic pore could be placed        inside a plasmonic structure such as a photonic nanocavity (see        FIGS. 5 and 6). The advantages of this method include the        well-characterised behavior of single molecules induced to pass        through such a pore, the small and highly reproducible pore        diameter an organic pore allows, and the low cost of producing        such organic pores.    -   The use of a solid state nanopore allows for control over the        pore diameter and for nanopositioning plasmonic structures such        that MEF or SERS is optimised. Ideally photon emission can be        further controlled by means of a waveguide. This would allow for        more efficient photon detection and higher detection speeds.    -   A third general method of construction is to allow for a random        distribution of highly localised EMFs such that the field        intensity at any given point on a porous surface is not known        and to allow for the single molecule to pass through this        surface at a controlled speed but uncontrolled location (FIG.        7). This has a tremendous advantage over the other two methods        in that as neither the location nor the distribution of either        the EMFs or the single molecules are precisely controlled, the        ease with which such a device can be constructed is        correspondingly greater. A method controlling the speed of the        single molecules at the intersection of a 20 nm pore and a        surface containing randomly distributed EMFs is described below.        This method has the advantage in that it is intrinsically        multiplexed. Even if the fluorescence produced by a single        molecule passing through an EMF is such that only one molecule        can be detected per second, the device can be multiplexed such        that several hundred or thousand or million pores are in        operation at once.        Method for Construction of an Apparatus According to the Present        Invention and Performing Sequencing

An apparatus according to the present invention comprises the following:

-   -   A first reservoir 19 capable of holding several μL of solution        and containing an electrode 26 embedded in its bottom surface;    -   A layer of silicon (substrate) 11 with an array of nanopores 10,        which are 20 nm in diameter and which are spaced 1 μm apart        etched in its surface. Electrodes 12 are embedded in the silicon        layer 11 such that each 20 nm diameter pore 10 contains an        electrode 12, and each electrode 12 is connected to a voltage        supply with a high skew rate (not shown);    -   A porous membrane 22, on which a regular array of gold        nanoparticles 14 are deposited, is bonded to the silicon layer.        The gold nanoparticles 14 are of known size and geometry, and        when excited at the appropriate frequency, produce an EMF;    -   A gel 24 such as agarose or polyacrylamide is applied to the        membrane 22 having an average pore size such that when ssDNA 20        is induced to migrate through the gel 22 it will adopt an        approximately linear conformation. This forms the control means        in the present embodiment of the invention;    -   A second reservoir 21 is provided on the other side of the        substrate 22 into which a molecule will pass after having        travelled through the pore;    -   An array of voltage modulators (not shown) with a high skew rate        are linked to a sensitive photon detector such as an avalanche        photodiode (not shown);    -   A photon detector such as an avalanche photodiode (not shown) is        provided;    -   A means of controlling an array of voltage supplies based on        input from a photon detector (not shown) is provided;    -   A laser of a frequency capable of inducing plasmon resonance in        the metal colloids of known size and geometry (not shown) is        provided.        Method of Operation to Perform Sequencing:

DNA is labelled via PCR as previously described such that all of twobases (C and A or G and T) in a given strand of ssDNA are fluorescentlylabelled with fluorescent labels which have a maximum emission near, butpreferably slightly red shifted in relation to, the plasmon resonantfrequency of the metal colloids 14 being used.

The labelled ssDNA is then placed in the first reservoir 19. Themembrane 22 coated with a nanoporous gel 24 and containing anapproximately even distribution of gold nanoparticles 14 such that thespacing between them varies randomly between 1 nm and 100 nm is placedabove the first reservoir 19. The silicon layer 11 is then placed on themembrane 22 and the entire assembly can be immersed in a conductingsolution. A positive charge is then given to the electrodes 12 insidethe 20 nm pores 10 such that ssDNA 20 will be induced to migrate throughthe second reservoir 21 into the gel 24, pass through the gel 24 inlinear conformation and pass the gold nanoparticle 14 array emittingphotons as they pass through the EMF produced by the plasmon resonance.As the fluorescently labelled random primers were used in the PCRreaction, the first photon emission will be produced by either the 5′ or3′ primer. The photon emission obtained from the primer will immediatelyindicate the speed at which the ssDNA 20 fragment can be induced tomigrate through the EMF and a clear signal still be obtained. As thepositioning of the ssDNA 20 in relation to the gold spheres 14 will bedifferent inside each of the 20 nm pores 10 for every ssDNA fragment 20,there will be an optimal voltage which can be applied to each pore 10 atany given time. As outlined above, a distance map containing thedistances relative to each other and to the known primers can be createdfrom the photon emission data. This data can then be assembled into acomplete genome sequence.

Additional Methods for Preparation of a Substrate and PerformingSequencing

There are many way in which a suitable substrate can be produced usingnanotechnological techniques. The following describes two possible waysand the manner in which a sequencing protocol would be preformed.

Example 1 Organic Pore in a Plasmonic Nanocavity (As illustrated inFIGS. 5 and 6)

1) An array of plasmonic structures such as photonic nanocavities 30 areconstructed of a suitable material 32 as described in [4], thenanocavities having an inside diameter of 8 to 10 nm as in FIG. 5, orhaving an inside diameter of 4 to 5 nm as in FIG. 6.

2) An organic pore 34 such as α-haemolysin is then positioned insidethese plasmonic structures by means suggested in [13] such that theorganic pores 34 are spaced approximately 1 μm apart.

3) The plasmon resonance wavelength of these nanocavities 30 is thenprecisely determined and a laser and fluorescent dye spectra are chosento correspond with the defined plasmon resonance. The fluorescent dyespectra should be slightly redshifted (20 to 150 meV) from the peak ofplasmon resonance.

4) Electrolytic transport through the organic nanopore 34 is carried outas described in [10].

5) Photon emission from the fluorescent labels is detected using anavalanche photodiode detection system well-known in the art.

6) After extracting DNA from a chosen genetic source, ssDNA isfluorescently labelled using PCR and random primers such as are used inwhole genome amplification. Of the four nucleotides present in the PCRreaction, one will be present only in its dye-modified form, ensuringhigh density fluorescent labelling of the chosen nucleotide. In order toensure high density labelling with long read lengths, severalfluorescent dyes may have to be tested with various polymerases asdescribed in [7].

7) This PCR reaction is then carried out for the remaining threenucleotides such that four PCR reactions will have been carried out,resulting in four samples containing DNA fluorescently labelled at allof the C, T, G, and A nucleotides respectively.

8) Immediately before the DNA is passed through the sequencing device,the PCR reactions are denatured as is well known in the art, to allowlabelled ssDNA to pass through the nanopores 34.

9) One denatured PCR reaction at a time is allowed to pass through thenanopore 34.

10) Photon emission from the dye molecules is obtained by an APDaccurate to one millionth of a second. The timings of these bursts ofphoton emission are normalised with what is known regardingtranslocation of ssDNA through a pore and several thousand or milliondistance maps are created revealing the spacing between identicalnucleotides.

11) These distance maps are then reassembled first into four completelyre-assembled sequences of identical nucleotides, and composited into acomplete genome sequence.

Example 2 Synthetic Nanopore (As illustrated in FIG. 7)

1) An array of conical nanopores having an inside radius at the tip of30 nm or less are etched in a semiconducting silicon nitride orconducting metal membrane 40 using electron beam lithography well knownin the art. These nanopores are spaced 1 μm apart. The etched siliconmembrane is installed on a stage as described in [11] and placed inelectroplating liquid in a small Teflon tube (2 mm diameter), whichoperates as an electroplating bath. TEMPEREX 8400 (Tanaka Kikinzoku)including 1.17% (w/w) KAu(CN)2 is used as the electroplating liquid atroom temperature. A gold wire (0.5 mm diameter) was immersed in thesolution as a counterelectrode. A bias voltage of 1.4 to 1.5 V is thenapplied to the initial electrodes relative to the counterelectrode inthe system. This is followed by the electrodeposition of gold on thesurface of the initial electrodes. Typically, an inducing current of 0.2mA allows an electroplating rate of 1 A°/s at room temperature.

2) A layer of gold 42 is then deposited using electrodeposition asdescribed in [11] to produce a pore 1.5 to 2.5 nm in diameter. Whenexcited by an incident wave of the appropriate wavelength, thisstructure will produce a highly localised EMF concentrated at the tip ofthe conical pore [12].

3) As the labelled ssDNA is induced to migrate through the pore 44 byelectrophoresis individual labels come into contact with the edge 43 ofthe pore 44. As the EMF is highly localised around the pore's 44 edge,as a fluorescent label passes through the field, photon emission wouldbe expected. Contact between the gold surface at the pore's 44 tip andthe fluorescently labelled ssDNA, however, induces fluorescentquenching. As the labelled ssDNA continues to move past the tip of thepore and into the channel, the channel expands. This expansioneliminates contact with the surface of the plasmonic structure and theresultant fluorescent quenching.

4) The plasmonic resonance wavelength of these electroplated conicalpores 44 is then precisely determined and a laser and fluorescent dyespectra are chosen to correspond with the defined plasmon resonance. Thefluorescent dye spectra should be slightly redshifted (20 to 150 meV)from the peak plasmon resonance.

5) Electrolytic transport through the conical nanopore 44 is carried outas described in [8,9].

6) Photon emission from the fluorescent labels is detected using anavalanche photodiode detection system well-known in the art.

7) After extracting DNA from a chosen genetic source, ssDNA isfluorescently labelled using PCR and random primers such as are used inwhole genome amplification. Of the four nucleotides present in the PCRreaction, one will be present only in its dye-modified form, ensuringhigh density fluorescent labelling of the chosen nucleotide. In order toensure high density labelling with long read lengths, severalfluorescent dyes may have to be tested with various polymerases asdescribed in [7].

8) This PCR reaction is then carried out for the remaining threenucleotides such that four PCR reactions will have been carried out,resulting in four samples containing DNA fluorescently labelled at allof the C, T, G, and A nucleotides respectively. 9) Immediately beforethe DNA is passed through the sequencing device, the PCR reactions aredenatured as is well known in the art, to allow labelled ssDNA to passthrough the nanopores 44.

10) One denatured PCR reaction at a time is allowed to pass through thenanopore 44.

11) Photon emission from the dye molecules is obtained by an APDaccurate to one millionth of a second. The timings of these bursts ofphoton emission are normalised with what is known regardingtranslocation of ssDNA through a pore 44 and several thousand or milliondistance maps are created revealing the spacing between identicalnucleotides.

12) These distance maps are then reassembled first into four completelyre-assembled sequences of identical nucleotides, and composited into acomplete genome sequence.

REFERENCES

[1] Fu, Yi, Lakowicz; Joseph R., “Single molecule studies of enhancedfluorescence on silver island films”, Plasmonics, Vol. 2, No. 1, 2007.

-   [2] Nakamura, Toshihiro; Hayashi, Shinji, “Enhancement of dye    fluorescence by gold nanoparticles: Analysis of Particle Size    Dependence”, Japanese Journal of Applied Physics, Vol. 44, No. 9A,    pp. 6833-6837, 2005.

[3] Pompa, P. P. et al, “Metal enhanced fluorescence of colloidalnanocrystals with nanoscale control”, Nature, published online 3 Nov.2006.

[4] Netti, M. Caterina et al, “Confined plasmons in gold photonicnanocavities”, Advanced Materials, Vol. 13, No. 18, 2001.

[5] Aizpurua, J. et al, “Optical properties of gold nanorings”, PhysicsReview Letters, Vol. 90, No. 5, 2003.

[6] Kucheyev, S. O. et al, “Surface-enhanced Raman scattering onnanoporous Au”, Applied Physics Letters, Vol. 89, 2006.

[7] Taurai, Tasara et al, “Incorporation of reporter molecule-labellednucleotides by DNA polymerases. II. High-density labelling of naturalDNA”, Nucleic Acids Research, Vol. 31, No. 10, 2003.

[8] Heng, J. B. et al, “The Electromechanics of DNA in a syntheticnanopore”, Biophysical Journal, Vol. 90, February 2006.

[9] Ho, Chuen et al, “Electrolytic transport through a syntheticnanometer-diameter Pore”, PNAS, Vol. 102, No. 30, 2005.

[10] Astier, Yann; Braha, Orit; Bayley, Hagan, “Towards single moleculeDNA sequencing”, Journal of American Chemical Society, Vol. 128, 2006.

[11] Kashimura, Yoshiaki et al, “Fabrication of nano-gap electrodesusing electroplating technique” Thin Solid Films 438-439, 2003.

[12] Downes, Andrew; Salter, Donald; Elfick, Alistair, “Simulations ofatomic resolution tip-enhanced optical microscopy” OPTICS EXPRESS, Vol.14, No. 23, 2006.

[13] Bayley, Hagan; Cremer, Paul S., “Stochastic sensors inspired bybiology”, NATURE, Vol. 413, September 2001.

The invention claimed is:
 1. An apparatus for investigating a molecule,the apparatus comprising: at least one nanopore provided in a substrate;a first reservoir suitable for receiving a sample including themolecule; a second reservoir separated from the first reservoir by thesubstrate; means to induce the molecule to move from the first reservoirto the second reservoir via the nanopore; at least one metallicnanostructure disposed around or adjacent to the nanopore facing thesecond reservoir, the metallic nanostructure producing, by particleplasmon resonance, a localized electromagnetic field around or adjacentto the nanopore facing the second reservoir; means to induce theparticle plasmon resonance in the metallic nanostructure; and means todetect fluorescence produced by the interaction of (i) the localizedelectromagnetic field produced by the metallic nanostructure disposedaround or adjacent to the nanopore facing the second reservoir and (ii)the molecule moving from the first reservoir to the second reservoir viathe nanopore and in close proximity to the metallic nanostructure. 2.The apparatus according to claim 1, wherein the nanopore has a diameterof from approximately 0.5 nm to 100 nm.
 3. The apparatus according toclaim 1, wherein the nanopore has a diameter of from approximately 1 nmto 50 nm.
 4. The apparatus according to claim 1, wherein the nanoporehas a diameter of from approximately 1 nm to 30 nm.
 5. The apparatusaccording to claim 1, wherein the nanopore has a diameter of from 1 nmto 5 nm.
 6. The apparatus according to claim 1, wherein the nanopore isorganic.
 7. The apparatus according to claim 1, wherein the substrate isa film.
 8. The apparatus according to claim 1, wherein the nanopore isformed from a nanoporous material selected from the group consisting ofporous gold, a colloidal crystal, a colloidal cavity and a metal toroid.9. The apparatus according to claim 1, wherein the nanopore is aplurality of nanopores passing through the substrate.
 10. The apparatusaccording to claim 9, wherein the plurality of nanopores are arranged inan array.
 11. The apparatus according to claim 1, wherein the metallicnanostructure comprises gold, silver, copper or aluminium.
 12. Theapparatus according to claim 1, wherein the metallic nanostructure issubstantially spherical.
 13. The apparatus according to claim 1, whereinthe metallic nanostructure is substantially annular.
 14. The apparatusaccording to claim 1, wherein the metallic nanostructure is a polygonalprism.
 15. The apparatus according to claim 1, wherein the metallicnanostructure has a diameter of from 50 nm to 150 nm.
 16. The apparatusaccording to claim 1, wherein the metallic nanostructure is disposedfacing the second reservoir so that the metallic nanostructure is 50 nmor less from the molecule as the molecule moves from the first reservoirto the second reservoir via the nanopore.
 17. The apparatus according toclaim 1, wherein the means to induce the molecule to move from the firstreservoir to the second reservoir via the nanopore comprises one or moreof means to induce electrophoresis, a micro-fluidics device, or a lasertrapping device.
 18. The apparatus according to claim 1, furthercomprising control means to control the movement of the molecule fromthe first reservoir to the second reservoir via the nanopore.
 19. Theapparatus according to claim 18, wherein the control means comprises aporous matrix through which the molecule passes, the porous matrix lyingimmediately adjacent to the entrance to the nanopore.
 20. The apparatusaccording to claim 1, wherein the means to detect fluorescence comprisesa photomultiplier or an avalanche photodiode.
 21. The apparatusaccording to claim 1, wherein the metallic nanostructure is a pair ofmetallic nanostructures, the localized electromagnetic field beingproduced, by the particle plasmon resonance, between the pair ofmetallic nanostructures, and wherein the pair of metallic nanostructuresare disposed around or adjacent to the nanopore facing the secondreservoir so that the molecule passes through the localizedelectromagnetic field being produced between the pair of metallicnanostructures as the molecule moves from the first reservoir to thesecond reservoir via the nanopore.
 22. The apparatus according to claim1, wherein the molecule is fluorescently labeled DNA or fluorescentlylabeled RNA.
 23. An apparatus for determining the location offluorescent labels in a nucleic acid comprised thereof, the apparatuscomprising: a substrate; a first reservoir suitable for receiving asample including the nucleic acid; a second reservoir separated from thefirst reservoir by the substrate; at least one nanopore provided in thesubstrate; at least one metallic nanostructure disposed around oradjacent to the nanopore facing the second reservoir, the metallicnanostructure producing, by particle plasmon resonance, a localizedelectromagnetic field around or adjacent to the nanopore facing thesecond reservoir; means to induce the nucleic acid to move from thefirst reservoir to the second reservoir via the nanopore; a photondetector for detecting photons arising from fluorescing of thefluorescent labels of the nucleic acid by the interaction of (i) thelocalized electromagnetic field produced by the metallic nanostructuredisposed around or adjacent to the nanopore facing the second reservoirand (ii) the nucleic acid moving from the first reservoir to the secondreservoir via the nanopore and in close proximity to the metallicnanostructure.
 24. The apparatus according to claim 23, wherein thenanopore has a diameter of from approximately 1 to 30 nm.
 25. Theapparatus according to claim 23, wherein the means to induce the nucleicacid to move from the first reservoir to the second reservoir via thenanopore comprises one or more electrodes.
 26. The apparatus accordingto claim 23, wherein the nanopore is organic.
 27. The apparatusaccording to claim 23, wherein the metallic nanostructure comprisesgold, silver, copper, or aluminium.
 28. The apparatus according to claim23, wherein the nanopore is a plurality of nanopores, and wherein theplurality of nanopores are arranged as an array in the substrate. 29.The apparatus according to claim 23, wherein the photon detector is aphotomultiplier or an avalanche photodiode.
 30. The apparatus accordingto claim 23, further comprising a laser for stimulating the particleplasmon resonance in the metallic nanostructure.
 31. The apparatusaccording to claim 23, wherein the sequence of nucleotides in thenucleic acid is determined from the detected photons arising from thefluorescing of the fluorescent labels of the nucleic acid by theinteraction of (i) the localized electromagnetic field produced by themetallic nanostructure disposed around or adjacent to the nanoporefacing the second reservoir and (ii) the nucleic acid moving from thefirst reservoir to the second reservoir via the nanopore and in closeproximity to the metallic nanostructure.
 32. The apparatus according toclaim 31, wherein the nucleic acid is DNA or RNA.
 33. The apparatusaccording to claim 23, wherein the metallic nanostructure is a pair ofmetallic nanostructures, the localized electromagnetic field beingproduced, by the particle plasmon resonance, between the pair ofmetallic nanostructures, and wherein the pair of metallic nanostructuresare disposed around or adjacent to the nanopore facing the secondreservoir so that the nucleic acid passes through the localizedelectromagnetic field being produced between the pair of metallicnanostructures as the nucleic acid moves from the first reservoir to thesecond reservoir via the nanopore.